ES2315205A1 - Bacillus amyloliquefaciens bam 2507 (cect 7313) depletioner of oil residues of the food and food industry, and procedure for its application. (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Bacillus amyloliquefaciens bam 2507 (cect 7313) bacterial strain, characterized by its capacity to degrade oily residues in the food and hospitality industry, due to its growth capacity in polyphenols-rich oily water and its biotransformation capacity of the polyphenols themselves and fats through their enzymatic activities esterase, esterase lipase and lipase. This strain has been isolated and characterized to be used in the biological treatment of oily effluents components of said residues, classified as toxic or dangerous, generated during the process of making olive oil and other vegetable oils, as well as after their subsequent consumption, according to a process that, for the particular case of the emulsions of edible oils used, consists of a prior physical-chemical treatment of the residue and the subsequent inoculation of the microorganism for biotransformation. (Machine-translation by Google Translate, not legally binding)

Description

Bacillus amyloliquefaciens Bam 2507 (CECT 7313) degrader of oily waste from the food and hospitality industry, and procedure for its application.

The object of the present invention is the bacterial strain Bacillus amyloliquefaciens , of laboratory code Bam 2507, isolated and characterized for the purpose of being used in the biodegradation of oily residues of the food and hospitality industry, as is the case of manufacturing and wholesale consumption of olive oil, due to the ability of said microorganism to develop in the waters and oily effluents rich in polyphenols components of said waste, classified as toxic or hazardous, and to degrade the polyphenols themselves and the existing fats in the same.

This strain has been deposited for the purpose of patent in the Spanish Type Culture Collection (CECT), dated  October 16, 2007, where it has been assigned the number 7313. The CECT is based in the research building of the University of Valencia, located on the Burjassot campus (DP 46100 - Valencia Spain).

The object of the present invention is also the procedure by which the microorganism is applied to treatment of those oily residues from consumption in hospitality of vegetable oil, mainly olive oil, with the end of the degradation process of oily emulsions It can really take place.

Therefore, the invention falls within the broad field of biotechnology, and within it, in the biological treatment of effluents and oily waters components of toxic or hazardous oil residues, both from the food industry, generated during the manufacturing process (known alpechines and alpeorujos in the case of olive oil), such as that produced after consumption in hospitality (edible oil emulsions
used).

For being a method of biological treatment of waste, is in itself an environmentally friendly method and economical, compared to traditional disposal or of physical-chemical treatments of waste Oily, but also in this case, to be directed to transform polyphenols and other especially toxic substances of the waters and effluents generated in the production and consumption of oils vegetables, it is a really effective method.

State of the art

Waste is defined as any substance or object from which its holder detaches or intends or obligation to detach (Law 10/1998 on waste). In agreement under current legislation, hazardous wastes are those that they meet at least one of the following characteristics: explosiveness, oxidizing, flammable, irritating, harmful, toxic, carcinogenic, corrosive, infectious, teratogenic, mutagenic, unstable, source of secondary or eco-toxic compounds. How example we can say that a residue is corrosive if it presents any of the following properties: be aqueous and have a pH less than 2 or more than 12.5, and / or be liquid and corrode the steel at a higher rate of 6.35 mm / year at a temperature of 55 ° C. And a residue would be toxic when it has the potential to cause death,  serious injuries, or detrimental effects on the health of the being human if ingested, inhaled or comes into contact with the skin.

As a result of certain activities related to vegetable oils, such as manufacture of olive oil, the hospitality sector, or the pig production, oily effluents are generated that includes substances toxic to health and harmful to the environment environment, so they are considered toxic waste or dangerous. These substances include the polyphenols

In the food and hospitality industry, the existing polyphenols are the result of many industrial processes related to vegetable oil, such as olive oil. In the effluents of olive oil production, both in oily waters or alpechines (extraction method continues in three phases), as in the pasty alpeorujos (extraction method in two phases), there is a high concentration of polyphenols, in addition of other organic components and inorganic compounds, all of a very toxic nature and in turn very diverse (Moreno et al ., 1990; Ramos et al ., 2000). In the particular case of polyphenols, most are considered extremely toxic to animal cells and pollutants to the environment, they also produce a very pronounced eutrophication action of water, which justifies the enormous contaminating effect of waste.

Therefore, the oily residues on which acts this patent proposal are considered as waste dangerous, both by the aforementioned state legislation (Law 10/98 of Waste), as per the Community (Council Directive 91/156). In the EU hazardous waste directive 91/689 / EEC in its Annexes I, II and III, classified into generic categories or types of related hazardous waste according to its nature or activity that generated them. So we have in Annex I.A. nº 9.- oil / water, hydrocarbon / water mixtures, emulsions; or in annex I.B. nº 19.- animal or vegetable soaps, fats and waxes; nº 38.- oils vegetables. We can also check this classification in the European Waste Catalog (CER), included in Chapter 20 of the list, corresponding to municipal waste (waste domestic and similar from commercial industries and institutions), including selectively collected fractions at point 20 01 25 and 20 01 26.

That is why the Waste Law obliges producers to eliminate (or transform) waste oily, either through an authorized manager, or through a treatment, such as an WWTP in the case of production of porcine. Its removal is a real problem for certain companies, especially if they are included in Annex 1 of the IPPC, as they are required to obtain Authorization Integrated Environmental

Traditional methods for elimination or treatment of effluents from the production of vegetable oils, especially regarding oily waters rich in polyphenols, have been direct elimination, through treatment plants, fertilizers, feed or controlled spills, the evaporation, natural in rafts or accelerated by heat treatment, and physical-chemical treatments (filtration, incineration, distillation-evaporation, etc.). These methods are generally harmful to the environment, and Also quite expensive. That's why a few years ago, to severely disrupted biological treatment methods, such as the generation of biogas, composting or fermentation, which is in the field where the invention falls below presents.

Legislation and technology oriented to the cleanliness of the environment and the prevention of its deterioration have been two of the greatest advances of the end of the twentieth century and have led to birth of technologies based on the use of microorganisms with broad biodegrading capabilities. In the particular case of the olive oil production industry there are antecedents that highlight the interest of treating biologically oily residues rich in polyphenols for compost production (FAIR CT96 1420. Annex A2 / Annex A2. FINAL REPORT. PROJECT IMPROLIVE Improvements of Treatments and Validation of the Liquid-Solid Waste from the Two-Phase Olive Oil Extraction. February 2000).

One such technique based on the use of microorganisms is bioremediation, which uses microorganisms live for the removal of environmental pollutants (Atlas and Pramer, 1990; Atlas and Bartha 2002). This is one of the technologies. applicable to the purpose pursued with the present invention.

For bioremediation to be effective, pollutants must be susceptible to microbial attack (metabolic transformation), metabolic products must be harmless and the process must not have adverse side effects on ecosystems. The environmental conditions must allow the in situ growth of the microbial agents that carry out the bioremediation or the extraction of the contaminant so that it can biodegrade ex situ in bioreactors.

There are many strains of microorganisms belonging to the genus Bacillus sp. that have been used in a multitude of biodegradation processes. Although some of the biotransformation routes of polyphenols by species of the genus Bacillus are known , we must consider that microbial species have an optimal degradation rate under particular conditions, and in the case of toxic or dangerous oils not yet biotransformation techniques are used with the express intention of reducing the concentration of polyphenols. A fact that supports this conclusion is the absence of patents or patent applications published by the SPTO in relation to biodegradation processes of oily waste from the food industry.

There is currently no work on biotransformation in toxic oils of industrial or hospitality origin published in patent documents or through non-patent literature, it is considered appropriate to propose more environmentally friendly disposal or reuse alternatives than those used until
moment.

The polyphenols found in aqueous waste generated in the production and use of vegetable oil, and which are a fundamental part of the oil fraction which is intended for consumption (Ortalli, 2004), they maintain, in general, a low degradation rate. In addition, this is only done by few species of microorganisms. That's why they are harder to eliminate naturally and persist longer affecting the environment. Among these polyphenols have been detected tyrosol, vanillic acid, protocatechic acid and caffeic acid. , and as more Toxic, catechol and methyl catechol.

What the inventor team has achieved is to isolate a bacterial strain of the genus Bacillus with degrading capacity of part of said toxic components, and which is especially novel with respect to strains of the same genus already known for their polyphenol degrading capacity, due to the ability to the new strain grow in an oily residue with high concentration of these substances and be able to degrade them at a high rate. In addition, the new microorganism has an enzymatic activity esterase, esterase lipase and lipase that allows it to degrade fats from oily residues of origin in the food and hospitality industry.

But this waste degrading microorganism Oils cannot be added directly to the residue, but requires a prior physical-chemical treatment of the same for use, which is also the object of this invention.

The invention

The aforementioned potentially degrading oily residue microorganism of the food and hospitality industry, and which is claimed in its own invention, is Bacillus amyloliquefaciens Bam 2507, with deposit number 7313 in the Spanish Type Culture Collection (CECT) for patent purposes .

It is a microorganism of the species B. amyloliquefaciens , genus Bacillus , with an important growth capacity in oily waters rich in polyphenols that are generated during the production process of vegetable oils, and which are also present in the emulsions of used oils from domestic consumption and hospitality, as well as the capacity of said microorganism for the biotransformation of polyphenols themselves and fats through their enzymatic activities esterase, esterase lipase and lipase existing in said waste.

The polyphenols of oily waters in which the microorganism in question develops and acts are tyrosol, vanillic acid, protocatechic acid, caffeic acid, catechol and methyl-catechol, among others.

Once isolated and characterized, it has been found that Bacillus amyloliquefaciens Bam 2507 has a particularly interesting biochemical activity for the purpose it is intended for. Among its features are the following:

It is a mesophilic microorganism, so the ideal incubation temperature should be between 28 and 40 degrees Celsius.

Also, the sporulated phase of the microorganism It is heat resistant, so it withstands temperatures below 80 ° C for long periods of time.

Maintains a metabolism aerobic-anaerobic facultative and is capable of biotransform the polyphenols in both conditions.

It behaves like tolerant acid but maintains an optimal biotransformation rate in pH of 5.5 to 7.

It has a fat-degrading activity through its enzymatic activities esterase, esterase lipase and lipase

Therefore, Bacillus amyloliquefaciens Bam 2507 can be used for its application in the elimination of polyphenols and fats from the oily waters of the food and hospitality industry, and in the biodegradation of oily residues of origin in this industry.

In cases where the oily residue of character  toxic or dangerous are made up of used vegetable oil emulsions (oily water with dissolved ionic charge), coming mostly volume of hospitality consumption, a Method of application of the bacterial strain of the invention for enable its biodegrading action of polyphenols and others toxic components once in contact with the residue.

This procedure, which is also claimed as of own invention, it consists of a treatment physical-chemical prior to waste and subsequent inoculation of the microorganism for biotransformation, according to these three basic operations described below in detail:

1) Emulsion of residual oil in decanter and separation of the aqueous-oily phase from the organic by water vapor flotation, performed by injection of water vapor from the bottom of the decanter.

2) Phase pH correction aqueous-oily up to a value between 5 and 7.5.

3) Inoculation of the microorganism in the phase aqueous-oily to cause its biotransformation

The advantage of this invention has become clear from all of the above; having a bacterial strain with the ability to degrade the polyphenols and fats present in the waters and oily effluents generated during the production and consumption of vegetable oils, such as olive oil, being the basis for a new biological treatment of these components residuals, a treatment that is clean and economical, as it is based on bioremediation processes, and especially effective as a decontaminant, when acting on high toxicity components, in reference to polyphenols, and on different types of fats that are very present in the
waste.

In addition, in the case of the treatment of waste of used vegetable oils, which are a problem in the hospitality sector, the procedure of application of the strain in question to the phase aqueous-oily of oily emulsions, which makes the invention directly usable in the industry.

The graph of Figure 1 reflects the efficacy of the invention as a decontaminant, when comparing the phenolic evolution of two oily media, one original, and another inoculated with Bacillus amyloliquefaciens Bam 2507, where it can be verified that while the polyphenol titration remains practically constant over time (V_ {0}, V_ {1}, V2}) in the original medium, in the inoculated with the bacteria decreases markedly, as explained below.

Application mode

The methodology for applying the Bacillus amyloliquefaciens Bam 2507 strain to oily residues of a toxic or dangerous nature has already been outlined above for a case where this residue is composed of emulsions of used vegetable oil (oily water with dissolved ionic charge) mainly from consumption in the hospitality industry.

As noted, the first is the preparation of the residue by treatment physicochemical, according to a process in which said emulsions are subjected to a steam flotation stage of water in order to separate the organic phase from the phase aqueous-oily, followed by the pH correction of This second.

The flotation operation is performed in a decanter in which steam is introduced through ducts with numerous injection nozzles 0.5 mm in diameter.

Water vapor injection from the bottom of the  decanter produces three effects: The first heating of the emulsion, thereby separating the organic phase from the aqueous one by increasing the buoyancy of oil with respect to water, the second it is the drag by flotation of the oil towards the surface, produced by the small ascending bubbles of steam, and by Finally, the solubilization capacity of acids is increased organic, ketones and aldehydes in the aqueous phase.

The organic phase can be used for others purposes such as biodiesel production, not being tested for the present invention. However, the phase aqueous-oily does not present this utility, being interesting to biotreat it with the intention of detoxifying it and being able to value it as compost or other uses.

The pH of the aqueous oil phase of the residue must be corrected until neutrality is approached before of the inoculation of the microorganism for its biotransformation. For that the process is optimal, the pH should be maintained between 5 to 7.5.

For pH correction it is proposed to use NaOH (sodium hydroxide) in concentration and volume according to amount and composition of the residue.

Once the residue is prepared, the inoculum of the Bacterial strain could be prepared from any general means complete and complex. The volume and concentration of the bacteria should adapt to the volume of the waste to be biotransformed, as well as the bioreactor to be used.

Inoculation of the microorganism in the phase aqueous-oily is made from a culture previous in liquid medium LB at 37 ° C for 24 h, until reaching a concentration greater than 5 x 10 11 cfu / ml (forming units of colonies per milliliter), verified by the counting method in plate, and inoculated at the rate of 500 ml of culture per cubic meter of residue.

Examples

The results of some of the tests and experiments that justify the efficacy of the invention

Table I shows the results of phenolic titration of an oily medium inoculated with the Bacillus amyloliquefaciens Bam 2507 strain in comparison to an original medium, where V1 is the polyphenol titration at t1 (mg / ml), V2 the polyphenol titration at t2 ( mg / ml), "Mean" is the statistical average of the corresponding value pair (mg / ml), and "D. standard", the standard deviation.

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TABLE I

one

The phenolic evolution of the two media compared, the original or inoculated with Bam 2507, is reflected in the graph of Figure 1. In this graph, V0 is the valuation of polyphenols at t0 (mg / ml), V1 polyphenol titration at t1 (mg / ml), with t1 being an incubation time of 7 days, V2 the titration of polyphenols at t2 (mg / ml), with t2 being a time of 14 day incubation.

Table II shows the assessment of the microbial activity esterase, esterase-lipase and lipase at 24 hours of Bacillus amyloliquefaciens Bam 2507, according to the API method ZYM BioMérieux.

TABLE II

2

The results confirm that the proposed strain for patent shows a marked esterase activity and esterase-lipase. Thus the fractionation of fats can facilitate the dissolution of atmospheric oxygen of the aerobic microbiota. This could be one of the keys that regulate the effectiveness of biotransformation in this process.

A last reuse of this waste (recovery) could be the biocompost being this strain a good candidate for this biotransformation for being present in a way normal on free ground. These microorganisms can favor the development and protection of crop species.

Generally, studies directed towards biodegradation have been carried out under anaerobic conditions in processes aimed at bioremediation (Bouallagui et al ., 2004), such as biodegradation of vegetable oils and their metabolic intermediates in freshwater sediments enriched with oil (Li et al ., 2005), but not exclusively. According to Eusebio et al . (2005), our study has been carried out under aerobic conditions, which may generate alternatives to the different biodegradation methods, especially those with treated effluents from the agri-food industry.

For Shuichi et al . (2001), composting is an anaerobic process of organic matter. The role of bacteria in compost depends on the variety of enzymes secreted by these microorganisms. The application of recombinant DNA techniques for transformation can increase the repertoire of digestive enzymes of these bacteria and will require information on their genetic systems. In this sense, the biocompost under anaerobic conditions has been used for the generation of methane as a source of energy (Bouallagui et al ., 2004). These emissions can also cause damage to the facilities and changes in the environment. However, this problem is minimized in aerobic conditions as is our case. In turn, the efficacy of Bacillus biotransformation is greater in aerobic conditions, which is why we set these conditions for our assay.

Claims (7)

1. Bacillus amyloliquefaciens Bam 2507 (CECT 7313), microorganism, genus Bacillus , characterized by its degrading capacity of oily residues of the food and hospitality industry, due to its growth capacity in oily water rich in polyphenols and its biotransformation capacity the polyphenols and fats themselves through their enzymatic activities esterase, esterase lipase and lipase. 2. Bacillus amyloliquefaciens Bam 2507 (CECT 7313), according to claim 1, wherein the polyphenols of the oily waters in which this microorganism develops and acts are tyrosol, vanillic acid, protocatechic acid, coffee acid, catechol and methyl catechol. 3. Use of Bacillus amyloliquefaciens Bam 2507 (CECT 7313), according to claims 1 and 2, in the biodegradation of oily waste from the food and hospitality industry. 4. Use of Bacillus amyloliquefaciens Bam 2507 (CECT 7313), according to claim 3, in the removal of polyphenols and fats from the oily waters of the food and hospitality industry. 5. Procedure for the application of the microorganism Bacillus amyloliquefaciens Bam 2507 (CECT 7313) to toxic or dangerous residues of used vegetable oils, for the purposes of the preceding claims, consisting of the following operations: 1) emulsion of oil in decanter and separation of the aqueous-oily phase of the organic by means of water vapor flotation, performed by injection of water vapor from the bottom of the decanter, 2) correction of the pH of the aqueous-oily phase to a value between 5 and 7.5, and 3) inoculation of the microorganism for biotransformation of the residue. 6. Procedure for the application of the microorganism Bacillus amyloliquefaciens Bam 2507 (CECT 7313) to toxic or dangerous residues of used vegetable oils, according to claim 5, characterized in that the correction of the pH of the aqueous-oily phase is carried out by adding sodium hydroxide ( NaOH), in concentration and variable volume depending on the quantity and composition of the residue. 7. Procedure for the application of the microorganism Bacillus amyloliquefaciens Bam 2507 (CECT 7313) to toxic or dangerous residues of used vegetable oils, according to claim 5, characterized in that the inoculation of the microorganism in the aqueous-oily phase is carried out from a previous culture in liquid medium LB at 37 ° C for 24 h, until reaching a concentration greater than 5 x 10 11 cfu / ml (colony forming units per milliliter), verified by the plate count method, and inoculated at a rate of 500 ml of culture per cubic meter of residue.